[丙型肝炎病毒非结构蛋白3在大肠杆菌中的诱导表达]
[Inducible expression of non-structural protein 3 of hepatitis C virus in E. coli].
作者信息
Cheng Jun, Zhong Yanwei, Liu Yan, Dong Jing, Yang Jizhen, Yang Shouchun
机构信息
Gene Therapy Research Center, Institute of Infectious Diseases,302 nd Hospital of PLA, Beijing 100039, China.
出版信息
Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2002 Mar;16(1):85-7.
BACKGROUND
To express recombinant non-structural protein 3 of hepatitis C virus (HCV) in E. coli.
METHODS
The non-structural 3 (NS3) region DNA fragment of HCV was amplified by polymerase chain reaction (PCR) and inserted into inducible proeukaryotic expressive vector pET 30C(+)at Bam H1/EcoR1 sites. The competent BL21 (DE3) E.coli was transformed, and then cultured and induced with IPTG. The expressed HCV NS3 protein was confirmed with ELISA and dot blot hybridization using HCV NS3-specific single chain Fv (ScFv) antibody.
RESULTS
1 893 bp DNA fragment of HCV NS3 coding region was amplified by PCR technique. HCV NS3 expressive vector pET-NS3 was constructed. After transformation with pET-NS3 and induction with IPTG, recombinant HCV NS3 protein was expressed and confirmed by specific ELISA and dot blot hybridization.
CONCLUSIONS
The recombinant HCV NS3 can be expressed in E. coli.
背景
在大肠杆菌中表达丙型肝炎病毒（HCV）的重组非结构蛋白3。
方法
通过聚合酶链反应（PCR）扩增HCV的非结构3（NS3）区DNA片段，并将其在Bam H1/EcoR1位点插入可诱导原核表达载体pET 30C(+)。转化感受态BL21(DE3)大肠杆菌，然后用异丙基-β-D-硫代半乳糖苷（IPTG）培养和诱导。使用HCV NS3特异性单链Fv（ScFv）抗体通过酶联免疫吸附测定（ELISA）和斑点杂交确认表达的HCV NS3蛋白。
结果
通过PCR技术扩增出1893 bp的HCV NS3编码区DNA片段。构建了HCV NS3表达载体pET-NS3。用pET-NS3转化并经IPTG诱导后，表达了重组HCV NS3蛋白，并通过特异性ELISA和斑点杂交进行了确认。
结论
重组HCV NS3可在大肠杆菌中表达。
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